Abstract
The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterol-rich lipoproteins from the circulation. Mutations to the receptor result in familial hypercholesterolemia, and over 60 of these occur in the calcium-binding epidermal growth factor-like domain pair. Two selected mutations in this region (G322S and R329P) were introduced into the domain pair and analyzed by in vitro refolding. Both exhibited differing levels of protein misfolding with R329P being the most pronounced. Solution NMR studies of the mutant domain pairs after purification established that a fraction of protein maintains a native-like fold and that this fraction contains two intact calcium-binding sites. An in vivo analysis of intact receptors containing these binding sites showed significantly reduced cell-surface expression compared with the native LDL receptor levels, again with R329P showing the most severe decrease. The sum of these results suggests that either local changes in structure or domain misfolding may be associated with the mutations. There is also the possibility that the misfolding of the calcium-binding epidermal growth factor-like pair region is propagated to other regions of the intact receptor, resulting in more global defects. Surprisingly, for both mutants, those full-length receptors that fold and reach the cell surface retain the ability to bind LDL and release the ligand upon exposure to low pH. This analysis provides significant insight into the protein defect resulting from each of the two mutations and allows their classification to be 2B (partially transport-defective). The results also highlight a range of misfolding defects that may be associated with familial hypercholesterolemia and may enable the prediction of the consequences of homologous disease-causing mutations to other proteins.
Highlights
The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterolrich lipoproteins from the circulation
The ligand-binding region is composed of a series of seven LDL receptor-type A (LA) modules followed by a region with a homology to the EGF precursor, an O-glycosylation region, a transmembrane region, and a cytoplasmic tail [11]
It was possible to predict the structural consequences of some misanion-exchange; EGF, epidermal growth factor; EGF-A, first EGF domain of the LDLR; EGF-B, second EGF domain of the LDLR; EGF-AB, EGF domain pair from LDLR; FH, familial hypercholesterolemia; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single-quantum correlation; LB medium, Luria-Bertani medium; LDLR, low-density lipoprotein receptor; RP, reverse-phase; WT, wild type
Summary
Expression Plasmids and Cell Lines—The plasmid construct for bacterial expression of the LDLR EGF-AB pair has been described previously [16]. A Poros Perfusion Chromatography R2/10 column (10/100 mm; Applied Biosystems) was used with an elution gradient of 2– 60% of a buffer containing 80% acetonitrile, 0.1% trifluoroacetic acid at a rate of 3.9%/ min using a flow rate of 8 ml/min prior to lyophilization The His tag was removed as described previously [16]. Eluted between 185 and 255 mM NaCl. eluted between 185 and 255 mM NaCl This material was collected and purified by RP-HPLC as described above using a Poros Perfusion Chromatography R2/10 column (4.6/100 mm) and an elution gradient of 2– 60% of a buffer containing 80% acetonitrile, 0.1% trifluoroacetic acid at a rate of 5.2%/min with a flow rate of 3 ml/min prior to lyophilization. DiI-LDL fluorescence was determined by flow cytometry as noted above
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